Differences in Ca²⁺ signaling underlie age-specific effects of secretagogues on colonic Cl⁻transport

Abstract

Taurodeoxycholic acid (TDC) stimulates Cl(-) transport in adult (AD), but not weanling (WN) and newborn (NB), rabbit colonic epithelial cells (colonocytes). The present study demonstrates that stimuli like neurotensin (NT) are also age specific and identifies the age-dependent signaling step. Bile acid actions are segment and bile acid specific. Thus although TDC and taurochenodeoxycholate stimulate Cl(-) transport in AD distal but not proximal colon, taurocholate has no effect in either segment. TDC increases intracellular Ca(2+) concentration ([Ca(2+)](i)) in AD, but not in WN and NB, colonocytes. In AD cells, TDC (5 min) action on Cl(-) transport needs intra- but not extracellular Ca(2+). NT, histamine, and bethanechol increase Cl(-) transport and [Ca(2+)](i) in AD, but not WN, distal colonocytes. However, A-23187 increased [Ca(2+)](i) and Cl(-) transport in all age groups, suggesting that Ca(2+)-sensitive Cl(-) transport is present from birth. Study of the proximal steps in Ca(2+) signaling revealed that NT, but not TDC, activates a GTP-binding protein, Galpha(q), in AD and WN cells. In addition, although WN and AD colonocytes had similar levels of phosphatidylinositol 4,5-bisphosphate, NT and TDC increased 1,4,5-inositol trisphosphate content only in AD cells. Nonresponsiveness of WN cells to Ca(2+)-dependent stimuli, therefore, is due to the absence of measurable phospholipase C activity. Thus delays in Ca(2+) signaling afford a crucial protective mechanism to meet the changing demands of the developing colon.

Lithocholic acid attenuates cAMP-dependent Cl− secretion in human colonic epithelial T84 cells

“…It is well recognized that primary (CDCA) and secondary (DCA) dihydroxy bile acids regulate epithelial ion transport processes and, in particular, stimulate colonic Cl Ϫ secretion (4,31,64,75). In contrast, neither the ␤-epimer of CDCA (UDCA) the primary trihydroxy bile acid (CA), nor the secondary monohydroxy bile acid (LCA) (4,41) (42) while inhibiting it in the rat colon (77).…”

“…Malfunctioning of one or more of the transporters and signaling proteins underlying this intricately regulated process can lead to disease (3). For example, ileal bile acid malabsorption leads to excess bile acids in the colon where it is well established that DCA and CDCA, but not UDCA, CA, or LCA, stimulate Cl Ϫ secretion leading to fluid loss and diarrhea (4,9,23,39,41,58,75).…”

Lubiprostone Activates Cl− Secretion via cAMP Signaling and Increases Membrane CFTR in the Human Colon Carcinoma Cell Line, T84

“…Cells were then lysed in a buffer containing in mM: EDTA 1, MgCl 2 2, b-mercaptoethanol 5, DTT 1, Tris-HCl 25, pH 7.4, and protease inhibitor cocktail. Cell membranes were isolated by centrifuge total lysate for 30 min at 100,000 9 g. SDS-PAGE and Western blotting were performed as described earlier [18]. Briefly, 50 lg protein from total lysate and membrane were loaded on 7.5% polyacrylamide gels to a PVDF membrane (Millipore, Billerica, MA) blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST), and were then incubated with anti-ClC-2 antibody (1:1000, Alomone Labs Ltd., Jerusalem, Israel) or anti-CFTR antibody (1:1000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in 1% nonfat milk in TBST overnight at 4°C.…”

Taurodeoxycholate Modulates Apical Cl−/OH− Exchange Activity in Caco2 Cells

“…In isolated, short-circuited rabbit colon, mucosal TCDC also decreased net sodium flux by 50% and abolished net chloride flux [14]. With respect to the modulation of chloride transport by bile acids, previous studies have extensively focused on examining the effects of bile acids on intestinal chloride secretion and have suggested that bile acids induce chloride secretion via the activation of various intracellular signaling cascades [4] such as cAMP-dependent kinase [15] and protein kinase C (PKC) [16] and increases in intracellular Ca 2+ [17][18][19].…”